Transfection of tagged constructs validate the expression and translation initiation site prediction of alternative proteins detected by LC-MS/MS.

<p>Top diagrams represent the constructs used to detect the co-expression of HA-tagged reference and GFP-tagged alternative proteins by western blot analyses of HeLa cell lysates. GFP is inserted before the alternative stop codon in frame with the AltORF. The black line represents a specific region of the endogenous mRNA. For AltORFs located in 5′UTRs of <i>ZNF83</i> and <i>SLC35A4</i>, the constructs do not contain the RefORF since the insertion of GFP may prevent the expression of the downstream RefORF. For AltORFs overlapping the 5′UTR and the RefORF (<i>IDH3B</i>), and for AltORFs overlapping the RefORF (<i>BDH2</i>, <i>NIPA1</i>, <i>SCARB2</i>), the HA tag was introduced before the GFP tag in frame with the RefORFs. Western blots show the co-expression of reference and corresponding alternative proteins in cell lysates with anti-HA and anti-GFP antibodies, respectively. The left and right lanes are cell lysates from cells expressing a construct with a normal alternative initiation AUG codon or with an inactivated alternative initiation AAG codon, respectively. NPTII, encoded in the expression plasmid, was used as a transfection control. Molecular weight markers in kDa are indicated on the right. Bottom panels show confocal/DIC images with the various cellular distributions of GFP-tagged alternative proteins. Nuclei were stained with Hoechst. Scale bar: 10 μm. * The reference protein was not detected due to the small size (<3 kDa) of the truncated HA-tagged reference IDH3B protein.